Chemical Basis of Prey Recognition in Thamnophiine Snakes: The Unexpected New Roles of Parvalbumins

Detecting and locating prey are key to predatory success within trophic chains. Predators use various signals through specialized visual, olfactory, auditory or tactile sensory systems to pinpoint their prey. Snakes chemically sense their prey through a highly developed auxiliary olfactory sense organ, the vomeronasal organ (VNO). In natricine snakes that are able to feed on land and water, the VNO plays a critical role in predatory behavior by detecting cues, known as vomodors, which are produced by their potential prey. However, the chemical nature of these cues remains unclear. Recently, we demonstrated that specific proteins–parvalbumins–present in the cutaneous mucus of the common frog (Rana temporaria) may be natural chemoattractive proteins for these snakes. Here, we show that parvalbumins and parvalbumin-like proteins, which are mainly intracellular, are physiologically present in the epidermal mucous cells and mucus of several frog and fish genera from both fresh and salt water. These proteins are located in many tissues and function as Ca2+ buffers. In addition, we clarified the intrinsic role of parvalbumins present in the cutaneous mucus of amphibians and fishes. We demonstrate that these Ca2+-binding proteins participate in innate bacterial defense mechanisms by means of calcium chelation. We show that these parvalbumins are chemoattractive for three different thamnophiine snakes, suggesting that these chemicals play a key role in their prey-recognition mechanism. Therefore, we suggest that recognition of parvalbumin-like proteins or other calcium-binding proteins by the VNO could be a generalized prey-recognition process in snakes. Detecting innate prey defense mechanism compounds may have driven the evolution of this predator-prey interaction

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Bioassays of the snake protein extracts derived from the cutaneous mucus of the prey species.

<p>The chemoattractivity of the crude mucus extracts was assessed according to a standard “all-or-none” snake bioassay based on a non-biological lure coated with the tested sample as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039560#pone.0039560-Leroy1" target="_blank">[9]</a>. The test was considered positive if the snake attacked the lure within 20 sec after the first lure-directed tongue flick. The test was considered negative in any other circumstances. A/B corresponds to the number of positive tests/total tests. X/Y corresponds to the number of positive tests/total tests after the proteinase K treatment.</p

Bioassays of the calcium-depleted or -supplemented <i>Osmerus eperlanus</i> parvalbumins.

<p>The chemoattractivity of the purified parvalbumins was assessed according to a standard “all-or-none” snake bioassay based on a non-biological lure coated with the tested sample as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039560#pone.0039560-Leroy1" target="_blank">[9]</a>. The test was considered positive if the snake attacked the lure within 20 sec after the first lure-directed tongue flick. The test was considered negative in any other circumstances. A/B corresponds to the number of positive tests/total tests. The purified form of <i>Osmerus eperlanus</i> α and β parvalbumins were obtained following the procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039560#pone.0039560-GosselinRey1" target="_blank">[33]</a>. The apo-parvalbumin was prepared by incubating the purified parvalbumin with a calcium-chelating agent (2 mM EDTA) and was then dialysed to remove the EDTA. ND  =  not determined.</p

The immunohistochemistry of the amphibian skin sections.

<p>(<b>A</b>) A <i>Lithobates catesbeianus</i> skin section stained with an anti-mucosal α parvalbumin (<i>Rana temporaria</i>) antibody. (B) A <i>Lithobates catesbeianus</i> skin section stained with an anti-muscular α parvalbumin (<i>Lithobates catesbeianus</i>) antibody. Note the localization of the immunoreactive cells in the dermal mucous glands (arrows). D  =  Dermis, E  =  Epidermis, MG  =  Mucous Glands.</p

A comparison of <i>E. coli</i> growth in the minimum culture medium (filled squares) in the presence of 12 mM EDTA (triangles) or 0.5 mM parvalbumins (squares).

<p>All of the results are shown as means ± s.e.m. for three independent replicate experiments per treatment. <i>Osmerus eperlanus</i> α parvalbumin was purified according to the procedure described by Gosselin and Rey (1977) (34). SDS-PAGE of purified <i>Osmerus eperlanus</i> α parvalbumin.</p

Parvalbumin threshold for eliciting prey-attack in <i>Nerodia fasciata</i>.

<p>The parvalbumin threshold for eliciting lure attack in <i>Nerodia fasciata</i> was assessed by using different quantities of purified parvalbumins in a bioassay. The threshold was shown to be in the tens of ug order of magnitude. Note that there is individual variation in the responses as individual 2 attacked lures coated with as low as 0.65 ug of parvalbumins. ND: response not determined.</p

Characterization of the crude mucosal extracts of (a) <i>Osmerus eperlanus,</i> (b) <i>Lithobates catesbeianus</i> and (c) <i>Pimephales promelas</i>.

<p>I : the protein extracts from cutaneous mucus were obtained from aqueous washes of the prey according to the procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039560#pone.0039560-Leroy1" target="_blank">[9]</a>. The filtered extracts were extensively dialysed against water containing 7 mM β-MSH and then lyophilized. The lyophilized mucus extract was solubilized in a Laemmli sample buffer and subjected to SDS-PAGE. The SDS-PAGE were stained with coomassie blue reagent II : The presence of parvalbumins was confirmed by rabbit anti-α parvalbumin western blot analysis of protein extracts obtained from the different cutaneous mucus. The migration of the immunoreactive products was consistent with the molecular weight of the parvalbumin family (10–14 kDa). No labeling was observed in the presence of the anti-α parvalbumin antibodies pre-incubated with the purified parvalbumins from the crude mucosal extract of <i>Lithobates catesbeianus</i> (E).</p

Bioassays of the immunoreactive SDS-PAGE bands extracted from the cutaneous mucus of the prey species.

<p>The chemoattractivity of the purified proteins was assessed according to a standard “all-or-none” snake bioassay based on a non-biological lure coated with the tested sample as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039560#pone.0039560-Leroy1" target="_blank">[9]</a>. The test was considered positive if the snake attacked the lure within 20 sec after the first lure-directed tongue flick. The test was considered negative in any other circumstances. A/B corresponds to the number of positive tests/total tests. ND  =  not determined.</p

Chemoattractivity of parvalbumins.

<p><b>A. The presence of calcium-complexed parvalbumins was monitored using SDS-PAGE. </b><i>Osmerus eperlanus</i> α parvalbumin was purified according to the procedure described by Gosselin and Rey (1977) (34). Apo-parvalbumin was prepared by incubating the purified parvalbumin with a calcium chelating agent (2 mM EDTA) and was then dialysed to remove the EDTA. As has been observed for <i>Rana temporaria</i> parvalbumins, a mass shift was observed in the absence of calcium after the electrophoresis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039560#pone.0039560-Leroy1" target="_blank">[9]</a>. <b>B. The purified α parvalbumin showed a chemoattractant activity.</b> A non-biological lure (cooked macaroni) was coated with 30 µl of protein sample or sample buffer (control solution) and placed in the snake’s box in front of its shelter. The test was considered positive if the snake attacked the lure within 20 sec after the first lure-directed tongue flick. In any other case, the test was considered negative. <b>C. The chemoattractant activity of proteins from crude mucus extract</b> The lyophilized mucus extract obtained from <i>Lithobates catesbeianus</i> was solubilized in a Laemmli sample buffer and subjected to SDS-PAGE (C.II). 13 pieces of gel were cut as described in the figure (C.I) and proteins from these excised gel pieces were recovered by pulverizing and soaking them in 50 mM DTE for 24 hours under strong agitation at 4°C. The supernatants were collected, lyophilized and assessed according to a standard “all-or-none” snake bioassay based on a non-biological lure coated as described above. – Négative test, + Positive test.</p